A presynaptic study of human versus insects sheds light on the composition and assembly of protein complexes in the insect synInsect comparative proteomes Abstract Background: The molecular components in synapses that are essential to the life cycle of synaptic vesicles are well characterized Nonetheless, many aspects of synaptic processes, in particular how they relate to complex behaviour, remain elusive The genomes of flies, mosquitoes, the honeybee and the beetle are now fully sequenced and span an evolutionary breadth of about 350 million years; this provides a unique opportunity to conduct a comparative genomics study of the synapse Results: We compiled a list of 120 gene prototypes that comprise the core of presynaptic structures in insects Insects lack several scaffolding proteins in the active zone, such as bassoon and piccollo, and the most abundant protein in the mammalian synaptic vesicle, namely synaptophysin The pattern of evolution of synaptic protein complexes is analyzed According to this analysis, the components of presynaptic complexes as well as proteins that take part in organelle biogenesis are tightly coordinated Most synaptic proteins are involved in rich protein interaction networks Overall, the number of interacting proteins and the degrees of sequence conservation between human and insects are closely correlated Such a correlation holds for exocytotic but not for endocytotic proteins Conclusion: This comparative study of human with insects sheds light on the composition and assembly of protein complexes in the synapse Specifically, the nature of the protein interaction graphs differentiate exocytotic from endocytotic proteins and suggest unique evolutionary constraints for each set General principles in the design of proteins of the presynaptic site can be inferred from a comparative study of human and insect genomes Background The completion of the Drosophila malengaster genome in the year 2000 provided the first glimpse at the make-up of animals with a complex nervous system [1,2] The availability of several genomes from insects, representing an evolutionary distance of 250 to 300 million years, provided a unique opportunity to evaluate the foundation of a functional synapse [3] With many additional animal genomes now Genome Biology 2008, 9:R27 http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, available, including those of primates, marsupials, fish and birds, a molecular correlation between genes and brain complexity is being actively sought [4,5] Drosophila has been used for decades as a model in which to study synapse formation, embryogenesis, development, and neurogenesis [6] A combination of biochemical, cell biologic, genetic, morphologic, and electrophysiologic studies have unravelled the molecular mechanisms of synaptic vesicle exocytosis and endocytosis in the fly [7,8] and compared these with the corresponding mechanisms in vertebrates [9] In all neurons, communication across the synapse is mediated by neurotransmitter release from synaptic vesicles Because the entire process may take only a fraction of a millisecond (in fast releasing synapses), additional processes ensure the priming, targeting, and docking of synaptic vesicles at the active zone [10] Only the basic mechanism of vesicle fusion is shared between yeast and human [11] Specifically, the minimal set of SNARE (Soluble NSF Attachment protein [SNAP] REceptor) functions is a unified mode of vesicle trafficking The proper targeting and docking of synaptic vesicles is mediated by a cognate interaction between vesicular SNAREs (v-SNAREs) and target membrane SNAREs (t-SNAREs) The genuine synaptic vesicle protein associated membrane protein (VAMP; also called synaptobrevin) acts as v-SNARE, whereas the presynaptic membrane proteins syntaxin and SNAP-25 (SNAP of 25 kDa) are t-SNAREs The multimeric ATPase NSF (N-ethylmaleimide sensitive fusion ATPase) is later recruited to the SNARE complex by SNAPs [12] and acts to break the extremely stable SNARE complex, thus reactivating the individual SNAREs for future fusion events Unlike yeast secretion and vesicle trafficking, synaptic vesicle fusion in the presynaptic structure requires a large body of regulators to ensure the spatial and temporal resolution of neurotransmitter release [13] Regulators of the SNAREs are numerous, and many of them are conserved throughout evolution Examples are the Rabs and their direct regulators [14] Specifically, Rab3, Rab5, Rab27, and Rab11 regulate vesicle transport, docking, and exocytosis of synaptic vesicles [15] Many of the other Rabs function in membrane trafficking in general and are strongly conserved [16,17] Recently, the composition and the stoichiometry of proteins and lipids of synaptic and transport vesicles from rat brain were presented [18] Based on Mass spectrometry (MS) proteomics technology, about 80 proteins were identified The synaptic role of many of these proteins was already established, mainly based on the genetics of model organisms such as Drosophila melanogaster and Caenorhabtidis elegans [2] Schematically, the proteins of the synaptic vesicles are associated with the following functional groups: organizers and cytoskeletal scaffold proteins; transporters and channels; Volume 9, Issue 2, Article R27 Yanay et al R27.2 sensors and signal transduction proteins; priming, docking, and fusion apparatus [19,20]; endocytotic and recycling machinery [7,21-23]; and linkers between the presynaptic and postsynaptic membranes [2] In addition, scaffolding proteins are critically important during the development and shaping of new synapses [24] These proteins are a combination of adhesion, cytoskeleton, and signaling proteins The specificity of neurons in the central nervous system (CNS) is primarily defined by the composition of receptors, transporters, and ion channels in the presynaptic and postsynaptic density (PSD) structures [25] In addition to their role in neuronal transmission through ion channels, PSD proteins are essential in establishing a protein network that bridges the cytoskeleton to the extracellular matrix [2] Herein, we focus on the basic function of the synapse, and specifically the trafficking, exocytosis, and endocytosis of synaptic vesicles, and analyze it in molecular terms We compiled a list of 120 gene prototypes, called 'PS120', which comprises the core set of proteins associated with synaptic vesicles and presynaptic structures This list includes components of the SNARE complex and their regulators, as well as components of the trafficking and organization apparatus of the active zone In comparison with humans, there are many fewer paralogous genes in the four insects whose genome sequence has been completed (namely fly, mosquito, honeybee, and beetle) This comparative view is instrumental for in silico genome annotations but it also exposes instances in which a specific gene or a regulation network is lost We show that the number of protein-protein interactions in which a protein participates and the degree of sequence conservation from insects to human are positively correlated The architectures of proteins responsible for processes in the synapse such as exocytosis and endocytosis differ markedly We show that a systematic comparative genomics view of the fly, honeybee, mosquito, and beetle proteomes reveals general principles in the design of presynaptic structures Results Evolutionary relationships among insects Insects are an ancient group of animals, the first of which probably appeared 360 to 400 million years ago Analyses of insect genomes and proteomes provide a unique opportunity to compare evolution between the model organism D melanogaster and numerous additional insect genomes The insects whose genomes were sequenced ensure coverage of a valuable phylogenetic breadth, spanning the fruit fly (D melanogaster(, the honey bee (Apis mellifera), the red flour beetle (Tribolium castaneum), the mosquitoes (Anopheles gambiae and Aedes aegypti), the silk worm (Bombyx mori) and the wasp (Nasonia vitripennis) All together, about 330,000 protein sequences from insects are currently available in public protein databases, which already include 12 additional Drosophila genomes A current list of insect Genome Biology 2008, 9:R27 http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, Volume 9, Issue 2, Article R27 Yanay et al R27.3 Table Presynaptic protein prototypes Number Gene Name S ADD2 β-Adducin D AMPH Amphiphysin M D AP2A1 AP-2 α-adaptin A AP3D1 AP-3 δ-adaptor A APBA1 Mint1 C APBA2 Adapter protein X11β B ARF1 ARF A ARF6 ARF A ARFGEF2 ARF-GEF B 10 ARFIP2 Arfaptin B 11 ATP6V0C ATPase 16 kDa A 12 BAIAP3 Bai1-associated D 13 BET1 Bet homolog B 14 BIN1 Bridging integrator D 15 BLOC1S1 Lysosome BLOC1 B * 16 BSN Bassoon E * 17 CACNA1A CaV2.1 B 18 CADPS Caps C 19 CALM2 Calmodulin A 20 CASK Lin-2 homolog B 21 CLTC Clathrin heavy chain A 22 CNO Cappuccino D 23 CNTNAP1 Neurexin D 24 CPLX2 Complexin C 25 DLG1 SAP 97 B 26 DNAJC5 HSP40 homologue B 27 DNM1 Dynamin A 28 DOC2B Double C2 EHD1 Testilin * * * * C 29 * A 30 EPN1 Epsin-1 C 31 EPS15 EGF substrate 15 D 32 ERC1 Rab6 interact CAST D 33 EXOC6 Exocyst C 34 EXPH5 Slp homolog E * 35 FLJ20366 Syntabulin E * 36 SNAP29 SNAP 29 D 37 GAP43 GAP 43 E 38 GDI2 Rab GDI B 39 GMRP P-selectin D 40 GOPC CFTR-associated ligand C 41 GOSR2 Membrin C 42 HGS Hepatocyte TK subs C 43 ITSN2 Intersectin D 44 KIF1A Kinesin family * B * 45 LAMP1 Lysosomal D 46 LIN7A Mals-1 A 47 LPHN1 α-Latrotoxin receptor D * 48 MSS4 Rabif C * Genome Biology 2008, 9:R27 http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, Volume 9, Issue 2, Article R27 Yanay et al R27.4 Table (Continued) Presynaptic protein prototypes 49 MUTED Muted D * 50 MYRIP Rab-Myosin 7A E * 51 NET2 Tetraspanin-12 C 52 NLGN2 Neuroligin-2 D 53 NRXN1 Neurexin D 54 NSF NEM-sensitive fusion B 55 PACSIN1 PKC and CK substrate C * 56 PCLO Piccolo D * 57 PICALM PI-binding clathrin C 58 PIK4CA P I4-kinase α C 59 PIP5K1C PI-4P 5-kinase 1γ B 60 PLDN Pallidin D 61 PPFIA3 Liprin α B 62 PSCD1 Cytohesin-1 A 63 PSCD2 Arno B 64 RAB27A Rab27A * B 65 RAB3A Rab3A A 66 RAB3GAP Rab3 GTPase D * 67 RAB3IL1 Rabin C * 68 RAB6IP1 Rab6 interacting C 69 RABAC1 YIP3 homolog C 70 RABGAP1 Rab GTPase C 71 RALA Ral A 72 RAPGEF4 Rap GEF C 73 SEC22B Sec22-like B 74 RILP Rab-interact E 75 RIMBP2 RimS binding * D 76 RIMS1 Rims D 77 RPH3A Rabphilin 3A C 78 SALF Stoned B * D C 79 SCAMP1 SCAMP37 80 SCIN Scinderin C 81 SEPT5 Septin B C * 82 SH3GL1 Endophilin 83 SIPA1L1 Signal-proliferation D 84 SLC17A7 VgluT1 C 85 SNAP25 SNAP-25 B 86 SNAP91 AP180 D 87 SNAPA SNAP B 88 SNAPAP Snapin C 89 SNIP Snip D * 90 SNPH Syntaphilin E * * 91 SNX9 Sorting nexin D 92 STX1A Syntaxin A 93 STXBP1 n-Sec B 94 STXBP5 Tomosyn C 95 STXBP6 Amisyn E 96 SV2A SV glycoprotein D 97 SYBL1 Synaptobrevin-like B 98 SYN Synapsin C * Genome Biology 2008, 9:R27 * http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, Volume 9, Issue 2, Article R27 Yanay et al R27.5 Table (Continued) Presynaptic protein prototypes 99 SYNGR1 Synaptogyrin C 100 SYNJ1 Synaptojanin C * 101 SYNPR Synaptoporin E * 102 SYP Synaptophysin E * 103 SYT1 Synaptotagmin B 104 SYT5 Synaptotagmin B 105 SYT9 Synaptotagmin C 106 SYTL4 Granulophilin C * 107 SYTL5 Synaptotagmin-like D * 108 TMEM163 synaptic vesicle31 E 109 TRAPPC1 Bet5 homolog C 110 TRAPPC4 Sybindin B * 111 TXLNA α-Taxilin C * 112 UNC13B Munc-13 B * 113 UNC13D Unc-13 homolog D 114 VAMP2 VAMP A 115 VAPA VAP33 C 116 VAT1 VAT-1 C 117 VPS18 Vacuolar sorting 18 D 118 VPS33B Vps-33B D 119 VTI1B Vti1 D 120 YWHAQ 14-3-3 protein * A The 120 presynaptic representatives from human (PS120) are indicated by their official gene names Sequence conservation between human and insect proteomes is indicated by A to E Sequence similarity index (S) is divided into five levels marked: A = >75%, B = >65%, C = >50%, D = >35%, and E = 75% throughout the sequence) for 16 genes This small set includes the v-SNARE VAMP2, the t-SNARE syntaxin 1A, and a few small GTP proteins (Ral, Rab3A, ARF1, and ARF6) In addition, this set includes essential components of the endocytic machinery (dynamin 1, AP2, AP3, EHD1, and clathrin) and proteins that activate transduction pathways (calmodulin and 14-3-3) That the function of these gene products is indispensable was expected, but proteins that coordinate synaptic vesicles with the active zone are also included in this selected list, namely cytohesin-1 [35] and Mals-1 [36] Both of these proteins share a function in determining the size of the readily releasable pool of synaptic vesicles and are critical for replenishing this pool In an attempt to gain new information on the structure and function of presynaptic proteins, we applied a comparative view and conducted multiple sequence alignment (MSA) analysis of human and insects for representatives of the exo- Figure sequence alignments Multiple1 (see following page) using for VAMP and synaptotagmin Multiple sequence alignments using for VAMP and synaptotagmin The multiple alignment sequence (MSA) is performed using ClustalW A graded blue color indicates the level of conservation among the representative sequences Horizontal line in the protein accessions separates insect (top) and vertebrate (bottom) sequences (a) Vehicle-associated membrane protein (VAMP; 11 sequences) The transmembrane domain is marked by a red frame Proline rich domain in the amino-terminal of mammalian VAMP-2 is framed in gray and was implicated in synaptophysin regulation Red arrows denote the identified tetanus toxin (X) and botulinum toxin (B, D, F, G) cleavage sites The star indicates an essential biogenesis targeting signal Stripped box indicates the calcium-calmodulin binding domain in mammalian VAMPs A conserved low complexity region that is shared among all insects is enriched with stretches of Ala, Gly and Pro, and is marked by a green frame Proteins (top to bottom): similar to CG17248 (iso A), honeybee; CG17248 (iso A), beetle; similar to VAMP, mosquito, CG17248 (iso A), honeybee; CG17248 (iso D), fruit fly; CG17248 (iso B), fruit fly; CG17248 (iso A), fruit fly; N-Syb, fruit fly, VAMP-2, human; VAMP-2, opossum; VAMP-1, human (b) Synaptotagmin (nine sequences) Calcium sensor for neurotransmitter release that is characterized by two C2 domains (marked in green frames) and an amino-terminal transmembrane domain (marked in an orange frame) Several interaction binding sites were located on synaptotagmin: tubulin (red stripped frame); calcium channels through syntaxin (gray stripped frame); and targeting signal to neurons that overlaps with the neurexin binding (blue stripped frame) Proteins (top to bottom): synaptotagmin, moth; CG3139 (iso A), beetle; synaptotagmin, mosquito; CG3139 (iso A), honeybee; CG3139 (iso C), fruit fly; CG3139 (iso A), fruit fly; CG3139 (iso A), fly obscura; synaptotagmin 1, human; synaptotagmin 1, opossum Genome Biology 2008, 9:R27 http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, Volume 9, Issue 2, Article R27 (a) F D VAMP X,B G honeybee beetle mos quito honeybee fruit fly fruit fly fruit fly fruit fly human opos s u m human (b) Synaptotagmin moth beetle mos quito honeybee fruit fly fruit fly fly obscura human opposum Figure (see legend on previous page) Genome Biology 2008, 9:R27 Yanay et al R27.7 http://genomebiology.com/2008/9/2/R27 Genome Biology 2008, Volume 9, Issue 2, Article R27 Yanay et al R27.8 cytotic machinery, VAMP-2, and synaptotagmin (Figure 1) VAMP-2 is a short, evolutionary conserved protein of 120 to 220 amino acids with a SNARE-interacting domain and a single transmembrane domain (TMD) that crosses the synaptic vesicle membrane Short signatures in VAMP's sequence that serve as recognition sites for tetanus and botulinum toxins [37] and the amino acids that are critical for VAMP targeting [38] are conserved from human to insects (Figure 1a) The sequence difference in the MSA is restricted to VAMP2 protein tails A short proline-rich region that is responsible for VAMP2 interaction with synaptophysin [39] is not conserved This is in accordance with the lack of synaptophysin in insect synaptic vesicles [40] (Table 1) On the other hand, a short region facing the synaptic vesicle lumen is highly conserved among all insects Interestingly, there are two VAMP variants in honeybee that differ only in their luminal domain, enforcing a functional difference between these two variants (Figure 1) The possibility that a functional binding domain is located in the luminal domain is consistent with findings for other synaptic vesicle proteins, including synaptotagmin [41] and SV2 [42] are suggested (syntaxin; Additional data file 5) These sequences are probably essential in interactions between yet undefined partners that are common to mammals and insects Most MSAs of PS120 show that the level of conservation is much higher among the insect sequences as compared with human or other organisms We emphasize that MSA from insects to human for strongly conserved proteins (synaptotagmin, syntaxin 1A, and VAMP2) and for much less conserved genes (stoned B, SCAMP1, and synapsin 1) is instrumental in detecting overlooked sequences that may be important for protein interactions, protein modifications, and regulatory functions The MSA for syntaxin and synapsin is included in Additional data file MSA of highly conserved sequences from human to insects was also performed for synaptotagmin (Figure 1b) Synaptotagmins belong to a large and diverse gene family that coordinate multiple signals with trafficking and with membrane fusion [5,43,44] In the mammalian synapse, synaptotagmin (and 2) is a genuine synaptic vesicle protein that serves as the calcium sensor and interacts with SNAREs as well as with the calcium channel [45] In addition, synaptotagmin is a linker to the endocytotic adaptor protein AP2 [46] The overall similarity of synaptotagmin between mammals and insects is high throughout the cytoplasmic region, but this similarity does not extend to the luminal region In the cytoplasmic region, the domain that was postulated to interact with AP2 and with neurexin is strongly conserved, suggesting that not only is the main function of the protein conserved but also is its engagement in a rich protein interaction network The exocyst is a large complex that was initially identified at the tip of the yeast bud It participates in tethering vesicles to the plasma membrane It coordinates exocytosis with small G-protein signalling molecules such as Ral-A, Arf6, and Rab11 [47] The exocyst is composed of eight subunits that are denoted EXOC1 to EXOC8 (Figure 2a) and are homologs of the yeast Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 genes [48] The level of conservation of the various subunits between human and fly range from 30% to 50% sequence identity (50% to 70% sequence similarity; Figure 2) The homologous relationship is evident and is supported by alignments that cover the entire protein length However, among the insects, the mutual sequence conservation for EXOC8 is rather low (Figure 2a), because the honeybee and beetle homologs for EXOC8 are further diverged; hence, an apparent homology could not be assigned Because the function of the exocyst relies on coordination of its subunits, we anticipated that EXOC8 would be missed during the task of genome annotation This is further supported by the observation that several interacting proteins of the exocyst such as Ral-A [47] and septin [49] are strongly conserved in all insects (Figure 2b) A search for sequence similarity in the honeybee and beetle genomes identified a supported mRNA for EXOC8 in honeybee and an apparently unprocessed sequence in the beetle genome (for details, see Additional data file 3) We conclude that physical complexes co-evolved because of similar evolutionary constraints Because endocytosis and membrane recycling are integral processes in presynaptic function, we compared stoned B (STNB) between human and insects [46] (Additional data file 4) Stoned genes (in insects StnA and StnB) are part of the protein lattice network that is involved in clathrin-mediated endocytosis at synapses The conservation level of human stoned B (called SALF) is rather low (